Biomaterials Lab 4 PPT Fall 2024 PDF

Biomaterials - ENGR-UH 4810 Fall 2024 Professor: Jeremy Teo Instructor: Shafiya Sabah 1 Lab 4: Fabrication of Collagen Hydrogel and its use as a Cell Culture System...

Biomaterials - ENGR-UH 4810 Fall 2024 Professor: Jeremy Teo Instructor: Shafiya Sabah 1 Lab 4: Fabrication of Collagen Hydrogel and its use as a Cell Culture System 2 Collagen and the Extracellular Matrix ▪ Collagen is the most abundant structural protein found in the extracellular matrix (ECM). ▪ ECM is a complex network of proteins and other macromolecules that surrounds cells in tissues. ▪ The ECM: ✓ Provides structural support for the cells ✓ Helps cells attach to and communicate with nearby The Extracellular Matrix cells ✓ Plays important role in cell growth and movement 3 Mimicking the Extracellular Matrix ▪ Mimicking the structural, mechanical, and physical properties of ECM through in- vitro tissue models is desirable to: ✓ Better understand cell behavior ✓ Study how changes in ECM contribute to disease progression ✓ Grow replacement tissue for regenerative medicine ▪ Due to their ability to mimic the biophysical and biochemical characteristics of native soft tissues, hydrogels are a highly attractive material for developing synthetic ECM analogs. 4 Mimicking the Extracellular Matrix ▪ Research has shown that cells in a 3D culture environment differ morphologically and physiologically from cells in a 2D culture environment. ▪ 3D cell culture systems represent more accurately the actual microenvironment found in tissues in the body. Cells in 2D and 3D microenvironments 5 Collagen Hydrogels ▪ Collagen hydrogels are being used as 3D cell culture substrates, as scaffolds in tissue engineering and to study tumor progression by reproducing the microenvironmental conditions of a solid tumor. ▪ Most collagen hydrogels are prepared using type I collagen, and is easily extracted from animal sources: bovine, porcine, rat tail tendon, and fish. Sources of Collagen 6 Collagen Hydrogels ▪ 3D collagen matrices with varying fiber thickness and length can be prepared in vitro by altering polymerization conditions such as: ✓ Collagen concentration ✓ pH ✓ Polymerization temperature 7 Collagen Hydrogels ▪ As concentration of collagen increases, the collagen fibers in the hydrogels become denser. ▪ As seen in the Figure, pore size decreases with high collagen concentration implying dense fiber network. Effect of collagen concentration 8 Collagen Hydrogels ▪ Increasing pH results in fibers with decreasing diameter. ▪ As seen in Figure, collagen fibers become thinner with increasing pH. Effect of pH 9 Collagen Hydrogels ▪ Collagen hydrogel polymerization occurs at a slow rate at low temperatures resulting in thicker fibers. ▪ The gel network acquires a more uniform diameter at polymerization temperature of 37 °C. ▪ Our experiment will involve use of rat tail type 1 collagen. ▪ The hydrogel polymerization will be initiated at physiological conditions of neutral pH (~ 7.4) and 37 °C. 10  Sequential images of the procedure to remove the skin and retrieve collagen fiber from a rat tail tendon Source: Rajan, N., Habermehl, J., Coté, MF. et al. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc 1, 2753–2758 (2006). https://doi.org/10.1038/nprot.2006.430 11 Lab Protocol ✓ Prepare a container with ice. ✓ Label 2 microcentrifuge tubes as 3 mg/ml and 1 mg/ml and place them on ice. ✓ Label 2 petri dishes as 3 mg/ml and , 1 mg/ml write your group number and place them on ice. ✓ Carefully place 3 coated coverslips on each petri dish. ✓ Add the following to the corresponding tubes in the same order as listed in the table: Coll (mg/ml) 3 1 0.1% Acetic Acid 0.000 0.526 Coll 0.789 0.263 Buffer 0.211 0.211 12 Lab Protocol ✓ Pipette the gel solutions onto coverslips of corresponding petri dishes such that each coverslip has 50 µL of solution. Working on ice is necessary to slow the polymerization. ✓ After pipetting, place the petri dishes in incubator set at 37 °C to form collagen hydrogels for 40-45 minutes. ✓ Take out the petri dishes, the gels appear turbid (white) indicating completion of gelation. 1 mg/ml collagen hydrogel 13 Lab Protocol Preparation of coverslips ▪ Glass coverslips are coated with APTES ((3- aminopropyl) triethoxysilane) and GA (glutaraldehyde). ▪ The APTES-GA coating is performed to immobilize hydrogels on the surface of the coverslip through covalent bonding. Coverslips placed in coating rack 14 15 16 17 18 (a) Rat tail collagen, (b) pH buffer and Acetic acid, (c) 1 mg/ml collagen hydrogel 19 RESULTS ✓ For 1 mg/ml collagen concentration, the hydrogel surface showed bigger pores as compared to 3 mg/ml collagen concentration. ✓ Highest fiber density was observed for 3 mg/ml collagen hydrogels. Surface of collagen hydrogel (a) 1 mg/ml (b) 3 mg/ml 20 RESULTS (a) Cell Nucleus and Cytoskeleton (1 mg/ml) (b) Cell Nucleus and Cytoskeleton (3 mg/ml) 21